Radiolabelling of proteins with fluorine-18 via click chemistry

Article abstract of DOI:10.1039/b916075b

The study describes for the first time the application of Cu(i)-mediated 1,3-dipolar [3+2]cycloaddition for the labelling of proteins with the short-lived positron emitter fluorine-18 as exemplified with azide-functionalized human serum albumin (HSA).

Full text of DOI:10.1039/b916075b

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Radiolabelling of proteins with fluorine-18 via click chemistry
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Theres Ramenda, Torsten Kniess, Ralf Bergmann, Jorg Steinbach and Frank Wuest*
Received (in Cambridge, UK) 5th August 2009, Accepted 20th October 2009
First published as an Advance Article on the web 4th November 2009
DOI: 10.1039/b916075b
The study describes for the first time the application of
Cu(I)-mediated 1,3-dipolar [3+2]cycloaddition for the labelling
of proteins with the short-lived positron emitter fluorine-18
as exemplified with azide-functionalized human serum albumin
(HSA).
chemistry was further applied for the design and synthesis of
radiometal-based radiotracers. Click chemistry was used to
form novel multidentate ligand scaffolds containing a triazole
group for the efficient chelation of a 99mTc(CO)3 core.17
However, to date, click chemistry has not yet been extended
to the radiolabelling of proteins with the short-lived positron
emitter 18F. In this work, we describe for the first time the
application of click chemistry for 18F labelling of proteins. The
reaction was accomplished through copper(I)-mediated
[3+2]cycloaddition between azide-functionalized human serum
albumin (HSA) and 4-[18F]fluoro-N-methyl-N-(prop-2-ynyl)-
benzenesulfonamide (p[18F]F-SA) [18F]3 as a novel click
chemistry building block with 18F on an aromatic carbon to
form a metabolically stable C–F bond.
Positron emission tomography (PET) is a powerful non-
invasive molecular imaging technique which provides functional
information on physiological, biochemical and pharmacological
processes in vivo.1–4 The steadily growing numbers of novel
molecular targets for PET imaging is accompanied with a
permanent demand for novel or improved radiolabelling
methods, especially for higher molecular weight compounds
like peptides, proteins, antibodies and antibody fragments. As
key regulators of cell growth and cellular function in living
organisms, radiolabelled peptides and proteins have been the
subject of intense research efforts for targeted diagnostic
imaging and radiotherapy in nuclear medicine over the last
30 years.
Sulfonamide 3 as reference compound and sulfonamide 6 as
a labelling precursor for 18F incorporation were prepared
starting from commercially available 4-fluorobenzene-1-sulfonyl
chloride or 4-nitrobenzene-1-sulfonyl chloride, respectively,
according to Fig. 1.
The short-lived positron emitter fluorine-18 (18F, t1/2
=
Acylation of sulfonyl chlorides with N-methylpropargyl
amine gave compounds 3 and 4 in excellent yields of 99%
and 96%. Reduction of nitro group in compound 4 with SnCl2
afforded the corresponding amine 5 in 62% chemical yield.
Treatment of amine 5 with an excess of MeOTf in the presence
of 2,6-di-tert-butyl-4-methylpyridine as the base provided
trimethyl ammonium triflate salt 6 as a labelling precursor in
excellent 97% yield suitable for subsequent radiolabelling
with cyclotron-produced [18F]fluoride according to a nucleo-
philic aromatic substitution reaction. The radiosynthesis
of 18F-labelled click chemistry building block [18F]3 was
accomplished in a single step in a remotely-controlled synthesis
apparatus (GE TRACERlabs FX-FN) using the powerful
nucleophilic radiofluorinating agent [18F]KF (generated by
treatment of cyclotron-produced [18F]fluoride with Kryptofix
109.8 min) is the radionuclide most frequently used for the
design and synthesis of PET radiotracers due to its favorable
nuclear and chemical properties. However, incorporation of
18F into high molecular weight compounds like proteins
represents a special challenge. Proteins cannot be labelled with
18F at high specific activity directly, due to the required
strongly basic reaction conditions at elevated temperatures,
which are not compatible with the structural and functional
integrity of proteins. To circumvent this obstacle, protein
labelling with 18F is usually accomplished by means of
prosthetic groups, also referred to as bifunctional labelling
agents,5,6 to allow 18F labelling under physiological conditions.
Linkage of 18F-labelled prosthetic groups to proteins can be
accomplished via various bioconjugation techniques, including
acylation,7–10 imidation,11 photochemical conjugation,8 and
thioether formation.12–14 However, every method has advantages
and limitations, and further work on the development of
rapid, clean and mild synthesis techniques for 18F-labelled
proteins is still needed.
K
222–potassium carbonate) in sulfolane as the solvent at 80 1C
for 10 min (Fig. 2). After HPLC purification, compound [18F]3
was isolated in 32 Æ 5% radiochemical yield (n = 11) at a
specific activity in the range of 120–570 GBq mmolÀ1. The
In recent years click chemistry has entered into many
fields of chemical sciences, including radiopharmaceutical
chemistry.15,16 The copper(I)-catalyzed 1,2,3-triazole formation
involving azides and terminal acetylenes according to a 1,3-
dipolar [3+2]cycloaddition has proved to be a particularly
valuable tool for efficient 18F labelling of peptides. Click
a Institute of Radiopharmacy, Research Center Dresden-Rossendorf,
Dresden, Germany
Fig. 1 Synthesis of reference compound 3 and labelling precursor 6.
(a) N-Methylpropargyl amine, CH2Cl2, 99% (3), 96% (4); (b) SnCl2,
EtOH, 62%; (c) MeOTf, 2,6-di-tert-butyl-4-methylpyridine, 97%.
b Department of Oncology, University of Alberta, Edmonton, Canada.
E-mail: wuest@ualberta.ca
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The 27 azide group-containing HSA was used for click
chemistry according to a 1,3-dipolar [3+2]cycloaddition with
18F-labelled alkyne [18F]3. Application of in situ reduction of
Cu(II) salts like CuSO4 into Cu(I) as required reactive copper
species within the copper-mediated [3+2] cycloaddition by
means of sodium ascorbate led to significant degradation of
HSA presumably due to partly or complete cleavage of the 17
disulfide bridges abundant in the protein.
Fig. 2 Radiosynthesis of click chemistry building block [18F]3.
radiochemical purity exceeded 95%. The use of other aprotic
polar solvents like DMF or DMSO gave lower radiochemical
yields.
In a typical experiment, starting from 16 GBq of [18F]fluoride,
6.5 GBq (dc) of click chemistry building block [18F]3 could be
obtained in a total synthesis time of 80 min, including HPLC
purification.
However, direct application of Cu(I) salts such as CuBr or
CuI would avoid the use of detrimental reducing agents like
sodium ascorbate. Various multidentate triazoles are known
as excellent chelates for stable complex formation of Cu(I)
preventing re-oxidation of Cu(I) into Cu(II).23,24 Therefore,
we used oligotriazole tris[(1-benzyl-1H-1,2,3-triazol-4-yl)-
methyl]-amine (TBTA) as a suitable chelator for stabilizing
Cu(I). Cu(I)–TBTA complex was applied to the click chemisty
reaction between azide-functionalized HSA and 18F-labelled
alkyne [18F]3. The reaction was performed in phosphate buffer
(pH 7.5) at 30 1C for 20 min. The product was purified by
SEC to give the desired 18F-labelled HSA25 in 55–60%
decay-corrected radiochemical yield based upon [18F]3. Fig. 4
shows the results of SEC and subsequent polyacrylamide gel
electrophoresis with sodium dodecylsulfate (SDS-PAGE) for
analysis of the reaction mixture and purified fractions. Up to
240 MBq of the final product were obtained within a total
synthesis time of 120 min starting from [18F]fluoride.
The convenient radiosynthesis of compound [18F]3 as a
novel 18F-labelled sulfonamide-based click chemistry building
block in a remotely-controlled synthesis unit allows its wide
application for a broad range of click chemistry reactions.
Moreover, the experimentally determined favorable lipophilicity
(log P = 1.7) of sulfonamide [18F]3 allows subsequent click
chemistry in aqueous media as relevant for proteins.
Human serum albumin (HSA) is the most abundant protein
in human blood plasma. HSA is composed of 585 amino acids,
and it has a molecular mass of 67 kDa. The amino acid
sequence contains 59 lysine residues which can be used for
the introduction of azide groups into HSA via reaction of
e-amino groups in the protein with an excess of 1-succinimidyl-
5-azidopentanoate.18,19 The modified protein20 was purified
with size-exclusion chromatography (SEC) to give 29% of
azide-containing HSA after lyophilization. MALDI-TOF MS
analysis of HSA and azide-functionalised HSA is given in
Fig. 3.
Small animal PET imaging with radiolabeled HSA in mice
was performed using a microPET_ P4 primate model scanner
(CTI Concorde Microsystems Inc., Knoxville, TN).26 Fig. 5
shows small animal PET images at 5 min, 60 min and 120 min
after radiotracer injection. After 120 min, most of the activity
was found in the gall bladder and intestines, and in the
bladder. The half-life of blood activity of the radiotracer was
calculated to be 31 min, which is faster compared with other
18F-labelled HSA derivatives reported in the literature.27
Hence, introduction of azide residues into HSA and subsequent
radiolabeling via click chemistry has significantly altered
structural and functional integrity of HSA.
The number of introduced azide residues was determined by
enzymatic digest of the azide-functionalized HSA followed by
MALDI-TOF MS analysis of the protein fragments according
to literature procedure.21 The enzymes trypsin, endoproteinase
Lys-C and endoproteinase Glu-C were used for enzymatic
digest. Each enzyme was used for
a single digest of
azide-modified HSA and native HSA as a control. The resulting
solutions were processed according to the delayed extraction
procedure22 prior to MALDI-TOF MS analysis. Seven
modified lysine residues could be detected after enzymatic
digest with trypsin and Glu-C, respectively, whereas 22 modified
lysine residues were determined after enzymatic digest with
Lys-C. Taking into account multiple detections of modified
lysine residues in the different enzymatic digests, the total
number of azide-modified lysine residues was determined
to be 27.
In summary, for the first time, click chemistry could
successfully be applied to the 18F labelling of proteins as
exemplified for azide-modified HSA. The reaction occurred
in a short reaction time compatible with the short half-life of
18F, and under mild reaction conditions compatible with the
Fig. 3 MALDI-TOF MS spectra of HSA (m/z = 66 184) and
azide-functionalised HSA (m/z = 69 705).
Fig. 4 SEC profile of the reaction mixture and SDS-PAGE analysis
of reaction mixture (lane 1) and purified fractions (lanes 2–5).
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Fig. 5 Small animal PET images of nude mice after intravenous
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20 Preparation of modified HSA. HSA (20 mg, 0.3 mmol) was disolved
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midyl-5-azidopentanoate (9 mg, 36 mmol) was dissolved in DMSO
(20 mL) and addedto the protein solution. The mixture was
incubated at room temperature for 2 h. The product was purified
by size exclusion chromatography by using a HiTrap desalting
column (5 mL, GE Healthcare Europe GmbH, Munich,
Germany). A subsequent dialysis step against water was performed
for 24 h at 4 1C (cutoff-value 14 000 Da). The resulting solution was
lyophilised and analysed by MALDI-TOF MS. The product was
obtained 29% isolated yield.
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structural and functional integrity of proteins. The application
of Cu(I)–TBTA complex provides a reliable and stable source
of Cu(I) for the click chemistry, and it circumvents in situ
reduction of Cu(II) salts as typically employed for Cu(I)-
mediated 1,3-dipolar [3+2]cycloaddition reaction. The successful
labelling of azide-functionalized HSA with the short-lived
positron emitter 18F according to Cu(I)-mediated 1,3-dipolar
[3+2]cycloaddition reaction further expands the scope of click
chemistry as a versatile tool for a broad array of radiolabelling
reactions.
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Chem. Commun., 2009, 7521–7523 | 7523